Alphaviruses are a group of arthropod-transmitted, enveloped, positive-strand RNA viruses classified in the Togaviridae family. The 30 plus members of the alphavirus genus differ significantly in the severity of disease induced in humans and other animals, from sub-clinical infections with Sindbis virus to fatal encephalitis that can be seen with eastern and Venezuelan equine encephalitis viruses. They have a lipid bilayer envelope and an icosahedral structure, and they are important human and animal pathogens. Our studies investigating the assembly pathway of alphaviruses have proven extremely fruitful in understanding the morphogenesis pathway and structure of this group of simple, yet important, enveloped viruses. Therefore, we propose to study alphavirus assembly using our previously successful approach, encompassing molecular genetics, protein and nucleic acid biochemistry, and structural biology. Our studies will build on very recent advances made in collaboration with our long-time colleague Michael Rossmann that have finally revealed the structure of the Sindbis virus E1-E2 spike using X-ray crystallography. The alphaviruses represent an excellent model system to understand how enveloped icosahedral viruses organize themselves into ordered arrays of proteins. The expertise of my colleagues at Purdue in protein and virus structure will complement our expertise in viral molecular genetics and biochemistry and maintain the dynamic environment in which these studies have, and will continue, to flourish. High resolution atomic structures of the alphavirus nucleocapsid protein and the envelope glycoproteins will be used in conjunction with pseudo-atomic resolution cryo-electron microscopy image reconstructions as a guide for the molecular genetic analysis of the nucleocapsid core and envelope proteins. We will rely heavily on the use of the large collection of biological reagents that we have produced and the expertise in genetics and structural biology accumulated during the previous period of this grant. In addition, we will use a variety of biophysical techniques that include X-ray crystallography, cryo-electron microscopy and electron tomography to probe the structure and assembly of both wild type and mutant viruses. The proposed experiments will focus on the spatial and temporal aspects of alphavirus assembly that guide oligomerization of the envelope glycoproteins into a spike arrangement, the association of the spikes with the pre-formed nucleocapsid core, and the incorporation of 6K protein and formation of the virus particle during budding. Our approach will be to incorporate recent and developing structural information together with genetic and cell biology approaches to develop a comprehensive understanding of virus assembly and egress. This insight will enable us, and others, to pursue novel strategies for virus and disease intervention.